The invention relates to a device for preparing specimens for an electron microscope, preferably a cryo-electron microscope, comprising a holder for holding a carrier, a movable blotting device for absorbing excess sample liquid applied on the carrier by means of a substantially planar absorbing medium, a control that is adapted to move the blotting device towards the carrier, and an environmental chamber in which the holder and the blotting device are arranged.
Further, the invention relates to a method for preparing specimens for an electron microscope, preferably a cryo-electron microscope in which the above-mentioned device is used, the method comprising the following steps: mounting a carrier in a holder, the holder being located in an environmental chamber, applying a sample liquid on the carrier for wetting the surface of the carrier with sample liquid, removing the excess sample liquid present on the carrier by means of an absorbing medium arranged on a movable blotting device, and absorbing the sample liquid into the absorbing medium, the blotting device being moved towards the carrier by means of a control for absorbing the sample liquid by the absorbing medium, and moving the blotting device away from the carrier by means of the control after termination of the absorption of excess sample liquid.
The performance of electron microscopy, in particular of transmission electron microscopy, in the study of the ultrastructure of biological specimens is remarkable. For meaningful examinations, however, a previous, structure-maintaining preparation of the specimen is necessary due to the high vacuum present in the electron microscope and the high-energy electron beam. Common preparation techniques mostly include a fixing of the sample by means of chemical fixing agents and an extraction of the water. In addition, for increasing the contrast an artificial contrasting by introducing atoms having a high atomic number is performed, e.g. by means of uranyl acetate in negative contrasting. All these preparation steps can result in deformations of the object to be examined or to the formation of artefacts, and require a careful and critical image analysis.
Cryo-electron microscopy has proven to be particularly suitable for examinations of biological structures. Given this technology, an aqueous sample is cryofixed, i.e. it is cooled very fast while avoiding the formation of ice crystals. The objects to be examined, for example cells, enzymes, viruses or lipid layers, are thus embedded in a thin vitrified ice layer and microscopically examined in this state. Cryofixed samples withstand the high vacuum present in the electron microscope. The great advantage of cryofixing over the afore-mentioned fixing methods is that the biological structures can be preserved in their native state and can be examined in their physiological environment. The risk of a formation of artefacts is significantly lower than is the case in the chemical preparation methods. However, the contrast is likewise very little and the low signal-to-noise ratio requires a reproduction of the structure information by means of image processing. Likewise disadvantageous is the higher sensitivity of the cryofixed samples to the electron beam. A contrasting is largely dispensed with due to the formation of artefacts, and the structure is digitally reconstructed.
Independent of the type of specimen preparation, it is inevitably necessary for a high-resolution transmission electron microscopic imaging that the specimen is sufficiently thin. Specimens for the transmission electron microscope are usually 30-100 nm, preferably 50-80 nm thick.
Specimens of this thickness can be obtained by cutting with the aid of an ultramicrotome, a sample embedded in polymer or a cryofixed sample (cryo-section) being cut into very thin sections. These so-called ultra-thin sections are then placed for microscopic examination on a carrier (also referred to as “grid”) that is common in electron microscopy. The ultra-thin section technique has the disadvantage that it is very complex and difficult to be automated. On the other hand, it allows “looking into” the inner live of structures, e.g. of cells or organelles, which were cut through in the middle.
Another preparation method refers to the application of thin liquid films on an electron microscopic carrier. In the preparation of cryo-electron microscopic specimens a thin liquid film is frozen very rapidly while preventing the formation of ice crystals. To this end, an electron microscopic carrier (“grid”) is immersed in a liquid containing the sample or the sample liquid is applied on the carrier by means of a pipette, the excess liquid is removed, for example, by means of a filter paper, and the liquid film remaining on the carrier is cryofixed by immersion in a bath of, for example, liquid ethane. In this context, the step of removing excess liquid, usually referred to as “blotting”, is decisive for the quality and the reproducibility of the liquid film. For cryo-electron microscopy a thickness of the liquid film of about 70 nm is to be aimed at. Further, it is desirable when the grid is coated with the liquid film as uniform as possible and with reproducibility. In addition, it is desirable to produce a large amount of specimens with reproducibility. Since in cryo-electron microscopy, a contrasting agent is mostly dispensed with, the images obtained are noisy. Therefore, structure data have to be reconstructed by means of digital image processing. In addition, the cryofixed specimens show a higher sensitivity to the electron beam and are quickly damaged. For obtaining high-resolution images, usually many similar images of identical objects are averaged. Cryo-electron microscopy thus makes particularly high demands on the reproducibility of the specimen preparations.
From WO 02/077612 A1 (see also EP 1 370 846 B1 and US 0200401572184) a device of the type mentioned at the beginning is known with which “blotting” can be performed almost automatically. This device is commercially available under the trade name “Vitrobot”. In this device, at first a carrier is fixed in a holder. After manual application of the sample liquid on the carrier, excess sample liquid is absorbed by means of one or two controllable blotting elements which can be moved towards the carrier (“blotting” operation). For the removing the liquid, a medium for absorbing liquid, e.g. a filter paper or another liquid-absorbing medium, is fixed on the blotting elements. The holder and the blotting elements are arranged in a humid environmental chamber in order to prevent drying out of the thin liquid film during the preparation. After blotting, the sample film is vitrified by immersion of the carrier in a cooling medium. Setting parameters comprise the number of blotting operations (a carrier can be blotted several times), the duration of the blotting, and the position of the carrier with respect to the filter paper. The device of WO 02/077612 A1 has the disadvantage that the blotting parameters are indeed reproducible, however the optimization requires long experimental series, e.g. when a sample liquid having a different viscosity is to be blotted. A further disadvantage is to be mentioned in connection with the described double-sided blotting mechanism. The carrier is squeezed by two filter paper disks on both sides with slight pressure. The pressure is dependent on the support for the filter paper, a foam rubber support, and can hardly be influenced. This can have a negative effect in particular for sensitive biological samples and coatings of the carrier grid and can result in mechanical damages to the objects to be microscopically examined or the delicate grid.
A semi-automated device similar to WO 02/077612 but having a simpler structure is commercially available under the trade name “Cryoplunge 3” by the company Gatan. Another device was described in the Article of S. Trachtenberg “A fast-freezing device with a retractable environmental chamber suitable for kinetic cryo-electron microscopy studies”, Journal of Structural Biology 123:45-55 (1998). Other devices do not have automatic or motorized blotting mechanisms, as a result whereof a reproducible thickness of the liquid film cannot be achieved. Blotting is a manual operation and dependent on the skill of the user.